Compositions and methods for preventing or treating cancer

ABSTRACT

The present invention relates to a MUC1 cytoplasmic tail peptide or portion thereof. These peptides are useful for inducing an immune response to MUC1-expressing tumor cells and thus for preventing or treating cancer.

This invention was made in the course of research sponsored by theNational Institutes of Health (NIH Grant Nos. CA72712, CA57362 CA09476).The U.S. government may have certain rights in this invention.

INTRODUCTION Background of the Invention

MUC1 is a large, transmembrane glycoprotein expressed on the apicalsurface of many types of polarized epithelia including pancreas, lung,breast and the gastrointestinal tract (Finn, et al. (1995) Immunol. Rev.145:61). MUC1 is overexpressed and differentially glycosylated by anumber of adenocarcinomas (Croce, et al. (1997) Anticancer Res. 17:4287)and has been evaluated as a candidate antigen for active immunotherapyprotocols. Humoral and cell-mediated immune responses against MUC1 aredetected in patients with MUC1⁺ tumors, as measured in vitro (Domenech,et al. (1995) J. Immunol. 155:4766; Petrarca, et al. (1999) CancerImmunol. Immunother. 47:272; Nakamura, et al. (1998) J. Gastroenterol.33:354); however, these responses are ineffective at eliminating thetumors in vivo.

A number of MUC1-based immunogens have been evaluated as potentialcancer vaccines (Graham, et al. (1996) Int. J. Cancer 65:664; Chien-Hungand Wu (1998) J. Biomed. Sci. 5:231; Reddish, et al. (1998) Int. J.Cancer 76:817; Heukamp, et al. (2002) J. Immunother. 25:46). Theseinclude whole cells expressing MUC1, MUC1 purified from tumor cells, andpeptide or glycopeptide fragments derived from the tandem repeat regionof MUC1 (Finn, et al. (1995) supra; Graham, et al. (1996) CancerImmunol. Immunother. 42:71; U.S. Pat. Nos. 5,744,144, 5,827,666 WO88/05054, U.S. Pat. Nos. 4,963,484 and 6,344,203). Clinical trials thatutilized MUC1 as a vaccine component focused on the tandem repeat region(Finn, et al. (1995) supra; Graham, et al. (1996) supra; Chien-Hung andWu (1998) supra; Reddish, et al. (1998) supra). Putative epitopes fromregions outside of the tandem repeat region of MUC1 have also beeninvestigated (Brossart, et al. (2000) Blood 96:3102; Brossart, et al.(1999) Blood 93:4309; Heukamp, et al. (2001) Int. J. Cancer 91:385);however, other potentially important epitopes from this tumor associatedantigen, especially those in the cytoplasmic tail, have not beenstudied. Most studies have used in vitro assays to investigate that thetandem repeat region contains immunodominant epitopes for production ofMUC1 specific antibodies and cytotoxic T-lymphocytes (CTL). However, ithas been shown that in vitro assays of cytolytic responses do notaccurately predict MUC1-specific tumor rejection (Tempero, et al. (1998)J. Immunol. 161:5500). For example, no detectable differences wereobserved in the anti-MUC1 CTL precursor frequencies of wild-type C57BL/6mice and C57BL/6 mice transgenic for human MUC1 (MUC1.Tg) (Tempero, etal. (1998) supra), although wild-type mice rejected MUC1-expressingtumors in a MUC1-specific manner while MUC1.Tg mice did not reject thesetumors and showed evidence of immunological tolerance to MUC1 (Tempero,et al. (1998) supra; Rowse, et al. (1998) Cancer Res. 58:315).

In vivo immune responses directed against tumor-associated MUC1 havealso been analyzed. The nature of cellular immune responses that mediaterejection of MUC1-expressing tumors in mice was investigated byexperiments that depleted CD4⁺, CD8⁺ or both T cell subsets in vivo.CD4⁺ cells were required for elimination of a human MUC1-expressingmurine melanoma cell line (B16.MUC1), and CD8⁺ cells were required forthe elimination of a human MUC1-expressing murine pancreatic carcinomacell line (Panc02.MUC1), in wild-type C57BL/6 mice (Tempero, et al.(1999) Int. J. Cancer 80:595; Morikane, et al. (2001) Int. Immunol.13:233). Studies using mice deficient in molecular components criticalto the immune responses (VanLith, et al. (2002) Int. Immunol. 14:873;Sivinski, et al. (2002) Cancer Immunol. Immunother. 51:327) furthershowed that both CD4⁺ and CD8⁺ responses were mediated by α/βT cellreceptors and required costimulation through CD28, as well asinteractions between CD40 and CD40 ligand, and the activities ofinterferon γ(IFNγ), and lymphotoxin α. A number of other factors (TL4,IL10, IL12, TNFR-1) were not required. There were differences in theeffector mechanisms as the CD8-mediated cytotoxicity required perforinbut not FasL; in contrast, the CD4-mediated cytotoxic response requiredboth perforin and FasL.

SUMMARY OF THE INVENTION

One aspect of the present invention is a MUC1 cytoplasmic tail peptideof SEQ ID NO:1 or a portion thereof for preventing or treating cancer ina subject. In a preferred embodiment, the MUC1 cytoplasmic tail peptideof SEQ ID NO:1 is part of a vaccine.

Another aspect of the present invention is a method for preventing ortreating cancer in a subject. The method involves administering to asubject an effective amount of a MUC1 cytoplasmic tail peptide of SEQ IDNO:1 or portion thereof so that cancer is prevented or treated in thesubject.

DETAILED DESCRIPTION OF THE INVENTION

Epitopes of MUC1 have now been found that are recognized and required bythe different MUC1 specific T cell populations (CD4 and CD8) mediatingthe antitumor responses. Putative epitopes were mapped by challengingmice with tumor cell lines (B16 and Panc02) that expressed constructs ofhuman MUC1 in which portions of the coding sequences for the proteinwere deleted. The C-terminus of the cytoplasmic tail (CT) and the tandemrepeat (TR) portion of MUC1 were required for rejection of B16.MUC1,while rejection of Panc02.MUC1 required a distinct portion of thecytoplasmic tail of MUC1, and not the tandem repeat. Vaccination withpeptides derived from the amino acid sequence of MUC1 cytoplasmic tailgenerated protective immune responses against MUC1-expressing tumors inMUC1.Tg mice. Survival of MUC1.Tg mice challenged with MUC1-expressingB16 or Panc02 tumor cells was significantly prolonged followingvaccination with three overlapping peptides spanning the entirecytoplasmic tail portion of MUC1. Further, vaccination with MUC1cytoplasmic tail peptides did not induce detectable autoimmuneresponses. These results demonstrate the importance of immunogenicepitopes outside of the tandem repeat of MUC1 and indicate thatimmunization with MUC1 cytoplasmic tail peptides is an effectiveanti-cancer immunotherapeutic approach.

Initially, the surface expression of human MUC1 variants encoded bydeletion constructs expressed in B16 and Panc02 cells was evaluated byflow cytometry. Control transfected B16 (B16.neo) and Panc02(Panc02.neo) were unreactive with the M2 or anti-MUC1 HMFG-2 antibodies.At least two representative clones of each cell line, with similar invitro growth rates, and expressing similar levels of the MUC1 isoforms,as determined by western blot analysis, were selected for furtherstudies.

Wild-type C57BL/6 mice produce MUC1-specific immune responses whenchallenged with MUC1-expressing B16 tumor cells (Tempero, et al. (1998)supra; Rowse, et al. (1998) supra), and Panc02 tumor cells (Morikane, etal. (2001) supra; Morikane, et al. (1999) Cancer Immunol. Immunother.47:287), which are lacking in MUC1.Tg mice because of immunologicaltolerance, as evidenced in vivo by differences in survival among thesestrains. It has now been shown that these immune responses in wild-typemice against B16.MUC1 and Panc02.MUC1 are MUC1-specific as the survivalof wild-type and MUC1.Tg mice challenged with B16.neo or Panc02.neocells were statistically indistinguishable (p>0.05). Immunodominantepitopes for MUC1-specific immune responses in vivo were identified bychallenging mice with tumors expressing recombinant isoforms of MUC1that lacked defined regions of the cytoplasmic tail or tandem repeat.Evidence that the deleted portion of MUC1 contributed to MUC1-specificimmune responses was obtained when survival curves for wild-type animalschallenged with B16 or Panc02 tumor cells expressing the deleted formswere similar to those of MUC1.Tg mice or mice challenged withMUC1-negative controls, B16.neo or Panc02.neo. If the deleted portion ofMUC1 was not required for MUC1-specific rejection of tumors, then thesurvival curves would have more closely resembled those of wild-typemice challenged with B16 or Panc02 cells expressing full-length MUC1.

The contribution of the cytoplasmic tail of MUC1 to MUC1-specific immuneresponses against MUC1 expressing B16 tumors was analyzed. Thecytoplasmic tail was examined for epitopes that contributed toMUC1-specific immune responses directed against B16.MUC1 tumor cells.Wild-type and MUC1.Tg mice were challenged subcutaneously with 2×10⁴ B16tumor cells expressing a construct in which all but three amino acids ofthe C-terminus were deleted (B16.MUC1.CT3). The removal of thecytoplasmic tail eliminated MUC1-specific immune responses towardB16.MUC1 (p>0.05), indicating that the cytoplasmic tail was critical tothe immunological rejection of these tumors (Table 1).

TABLE 1 Difference in Median Survival Tumor Cell Type (Wildtype −MUC1.Tg) p Value B16.MUC1 14.00 Days <0.0001 B16.neo −1.00 Days 0.518B16.MUC1.CT3 0.00 Days 0.322 B16.MUC1.CT33 1.00 Days 0.100 B16.MUC1.CT45−1.50 Days 0.253 B16.MUC1(ΔTR) 0.00 Days 0.231 P values were determinedfrom Kaplan-Meier survival curves.

Immune responses to two additional constructs expressed by B16 tumorcells were examined. In these constructs the final 36 amino acids(MUC1.CT33) or the final 24 amino acids (MUC1.CT45) of the cytoplasmictail were deleted. MUC1-specific tumor rejection was not detected inwild-type mice challenged with B16 tumor cells that expressed eitherMUC1.CT33 (p>0.05) or MUC1.CT45 (p>0.05). These results indicated thatthe final 24 amino acid segment of the cytoplasmic tail contained anepitope or region that was critical for immunological responses thatmediated rejection of MUC1-expressing B16 tumor cells.

The contribution of the tandem repeat to MUC1-specific immune responsesdirected against B16.MUC1 tumor cells was similarly evaluated. Wild-typeand MUC1.Tg mice were challenged subcutaneously with 2×10⁴ B16 cellsexpressing a construct in which the large, extracellular tandem repeatportion was deleted (B16.MUC1 (ΔTR)). Wild-type mice challenged withB16.MUC1 (ΔTR) experienced survival similar to MUC1.Tg mice challengedwith this tumor cell line (p>0.05) These findings indicate that thetandem repeat was an important immunological target during rejection ofB16.MUC1 tumor cells, when expressed together with the final 24 aminoacid segment of the cytoplasmic tail.

In parallel studies, the contribution of the cytoplasmic tail toMUC1-specific immune responses against MUC1-expressing Panc02 tumorcells was determined. It has been demonstrated that wild-type C57BL/6mice produce cell-mediated, MUC1-specific immune responses that rejectMUC1-expressing Panc02 tumors, whereas MUC1.Tg mice do not reject thesetumors (Morikane, et al. (2001) supra; Morikane, et al. (1999) supra).Groups of wild-type and MUC1.Tg mice were challenged subcutaneously with1×10⁶ Panc02 tumor cells expressing MUC1.CT3. Similar to results usingB16.MUC1.CT3 tumor cells, the removal of the cytoplasmic tail eliminatedMUC1-specific immune responses toward Panc02.MUC1 (p>0.05) (Table 2).

TABLE 2 Difference in Median Survival Tumor Cell Type (Wildtype −MUC1.Tg) p Value Panc02.MUC1 >34.50 Days 0.01 Panc02.neo −4.00 Days 0.38Panc02.MUC1.CT3 3.00 Days 0.44 Panc02.MUC1.CT33 29.00 Days 0.0056Panc02.MUC1.CT45 18.00 Days <0.0001 Panc02.MUC1(ΔTR) 8.00 Days 0.0058 Pvalues were determined from Kaplan-Meier survival curves.

Further analysis of the cytoplasmic tail to MUC1-specific immuneresponses against MUC1-expressing Panc02 tumor cells was conducted. Thesurvival of wild-type and MUC1.Tg mice following challenge with 1×10⁶Panc02 tumor cells expressing MUC1.CT33 was determined. In contrast toresults with B16 tumor cells expressing this form of MUC1, wild-typemice rejected these tumors and MUC1.Tg mice did not reject these tumors(p<0.05).

The survival of wild-type and MUC1.Tg mice following challenge with1×10⁶ Panc02 tumor cells expressing MUC1.CT45 was determined. Thewild-type mice rejected MUC1.CT45-expressing tumors in a MUC1-specificmanner, whereas MUC1.Tg mice did not (p<0.05). These findings indicatedthat an epitope or region between amino acid residues three and 33 ofthe cytoplasmic tail contributed to immunological responses necessary toreject MUC1-expressing Panc02 tumors.

The contribution of the tandem repeat to MUC1-specific immune responsesto Panc02.MUC1 was determined. Wild-type and MUC1.Tg mice werechallenged with 1×10⁶ Panc02 tumor cells expressing MUC1 (ΔTR).Prolonged survival was observed for wild-type mice compared to MUC1.Tgmice following challenge with Panc02.MUC1 (ΔTR) tumor cells (p<0.05).These results indicated that the tandem repeat portion of MUC1 was notrequired to reject Panc02.MUC1 tumor cells in a MUC1-specific manner.

Results provided herein demonstrate that epitopes in the MUC1cytoplasmic tail are critical for the immunological rejection of twodistinct MUC1-expressing tumors. Accordingly, the MUC1 cytoplasmic tailamino acid sequence was analyzed using two independent web-basedalgorithms that predict potential binding to major histocompatibilitycomplex (MHC) class I- and MHC class II molecules. Epitope predictionwas conducted for the 72 amino acid residue sequence of MUC1 cytoplasmictail:Cys-Gln-Cys-Arg-Arg-Lys-Asn-Tyr-Gly-Gln-Leu-Asp-Ile-Phe-Pro-Ala-Arg-Asp-Thr-Tyr-His-Pro-Met-Ser-Glu-Tyr-Pro-Thr-Tyr-His-Thr-His-Gly-Arg-Tyr-Val-Pro-Pro-Ser-Ser-Thr-Asp-Arg-Ser-Pro-Tyr-Glu-Lys-Val-Ser-Ala-Gly-Asn-Gly-Gly-Ser-Ser-Leu-Ser-Tyr-Thr-Asn-Pro-Ala-Val-Ala-Ala-Ala-Ser-Ala-Asn-Leu(SEQ ID NO:1). Multiple peptides within the regions required forrejection of Panc02.MUC1 (amino acids 3-33) and B16.MUC1 (amino acids45-69) were predicted to exhibit high binding affinity for murine MHCclass I. The results for binding to K^(b) and D^(b) are summarized inTable 3.

TABLE 3 Predicted H-2 SYFPEITHI BIMAS Epitope Location Molecule Score¹Score² Cys-Arg-Arg-Lys- 3 H2-D^(b) 20 10.171 Asn-Tyr-Gly-Gln- Leu (SEQID NO: 2) Val-Ser-Ala-Gly- 49 H2-D^(b) 14 No Score Asn-Gly-Gly-Ser-Obtained Ser (SEQ ID NO: 3) Leu-Ser-Tyr-Thr- 58 H2-D^(b) 13 33.480Asn-Pro-Ala-Val- Ala (SEQ ID NO: 4) Ser-Ala-Gly-Asn- 50 H2-D^(b) 1822.176 Gly-Gly-Ser-Ser- Leu (SEQ ID NO: 5) Ala-Val-Ala-Ala- 64 H2-D^(b)12 10.088 Ala-Ser-Ala-Asn- Leu (SEQ ID NO: 6) Val-Ser-Ala-Gly- 49H2-D^(b) 25 Decamer Asn-Gly-Gly-Ser- Option Ser-Leu N/A (SEQ ID NO: 7)Pro-Ala-Val-Ala- 63 H2-D^(b) 15 Decamer Ala-Ala-Ser-Ala- Option Asn-LeuN/A (SEQ ID NO: 8) Leu-Ser-Tyr-Thr- 58 H2-D^(b) 14 DecamerAsn-Pro-Ala-Val- Option Ala-Ala N/A (SEQ ID NO: 9) Phe-Pro-Ala-Arg- 14H2-D^(b) 13 Decamer Asp-Thr-Tyr-His- Option Pro-Met N/A (SEQ ID NO: 10)Arg-Arg-Lys-Asn- 4 H2-K^(b) 22 Octamer Tyr-Gly-Gln-Leu Option (SEQ IDNO: 11) N/A Asp-Arg-Ser-Pro- 42 H2-K^(b) 18 Octamer Tyr-Glu-Lys-ValOption (SEQ ID NO: 12) N/A Ala-Arg-Asp-Thr- 16 H2-K^(b) 17 OctamerTyr-His-Pro-Met Option (SEQ ID NO: 13) N/A Ser-Ser-Leu-Ser- 56 H2-K^(b)14 Octamer Tyr-Thr-Asn-Pro Option (SEQ ID NO: 14) N/A Cys-Arg-Arg-Lys- 3H2-K^(b) No Score 1.440 Asn-Tyr-Gly-Gln- Obtained Leu (SEQ ID NO: 15)Ala-Val-Ala-Ala- 64 H2-K^(b) No Score 1.210 Ala-Ser-Ala-Asn- ObtainedLeu (SEQ ID NO: 6) Ser-Ala-Gly-Asn- 50 H2-K^(b) No Score 1.100Gly-Gly-Ser-Ser- Obtained Leu (SEQ ID NO: 5) Ser-Glu-Tyr-Pro- 24H2-K^(b) No Score 1.100 Thr-Tyr-His-Thr- Obtained His (SEQ ID NO: 16)Location is the position of the first residue. ¹Score obtained usingprogram developed by Rammensee, et al. (1999) Immunogenetics 50: 213.²Score obtained using program developed by Parker, et al. (1994) J.Immunol. 152: 163. Score for H2-D^(b) obtained using D^(b) revised. N/Ais not available.

Similar analysis revealed numerous putative epitopes within theseregions predicted to bind human HLA molecules (Table 4).

TABLE 4 Predicted Loca- HLA SYFPEITHI BIMAS Epitope tion MoleculeScore^(1,3) Score^(2,3) Ser-Leu-Ser-Tyr- 57 HLA- 23 69.552Thr-Asn-Pro-Ala- A*0201 Val (SEQ ID NO: 17) Ser-Ala-Gly-Asn- 50 HLA- 200.297 Gly-Gly-Ser-Ser- A*0201 Leu (SEQ ID NO: 5) Tyr-Thr-Asn-Pro- 60HLA- 20 0.730 Ala-Val-Ala-Ala- A*0201 Ala (SEQ ID NO: 18)Ala-Val-Ala-Ala- 64 HLA 20 1.869 Ala-Ser-Ala-Asn- A*0201 Leu (SEQ ID NO:6) Ser-Tyr-Thr-Asn- 59 HLA- 27 HLA Type Pro-Ala-Val-Ala- A*0203 N/AAla-Ala (SEQ ID NO: 19) Asp-Thr-Tyr-His- 18 HLA-A1 22 1.250Pro-Met-Ser-Glu- Tyr (SEQ ID NO: 20) Gly-Asn-Gly-Gly- 52 HLA-A1 22 0.625Ser-Ser-Leu-Ser- Tyr (SEQ ID NO: 21) Pro-Thr-Tyr-His- 27 HLA-A1 21 0.125Thr-His-Gly-Arg- Tyr (SEQ ID NO: 22) Asp-Ile-Phe-Pro- 12 HLA-A1 20 5.000Ala-Arg-Asp-Thr- Tyr (SEQ ID NO: 23) Pro-Ser-Ser-Thr- 38 HLA-A1 20 0.075Asp-Arg-Ser-Pro- Tyr (SEQ ID NO: 24) Ala-Gly-Asn-Gly- 51 HLA-A1 23 NoScore Gly-Ser-Ser-Leu- Obtained Ser-Tyr (SEQ ID NO: 25) Ser-Thr-Asp-Arg-40 HLA-A1 20 No Score Ser-Pro-Tyr-Glu- Obtained Lys-Val (SEQ ID NO: 26)Asp-Thr-Tyr-His- 18 HLA-A26 32 HLA Type Pro-Met-Ser-Glu- N/A Tyr (SEQ IDN0: 20) Asp-Ile-Phe-Pro- 12 HLA-A26 30 HLA Type Ala-Arg-Asp-Thr- N/A Tyr(SEQ ID NO: 23) Asp-Ile-Phe-Pro- 12 HLA-A26 20 HLA Type Ala-Arg-Asp-Thr-N/A Tyr-His (SEQ ID NO: 27) Asp-Ile-Phe-Pro- 12 HLA-A3 22 0.900Ala-Arg-Asp-Thr- Tyr (SEQ ID NO: 23) Lys-Val-Ser-Ala- 48 HLA-A3 21 NoScore Gly-Asn-Gly-Gly- Obtained Ser-Ser (SEQ ID NO: 28) Tyr-Val-Pro-Pro-35 HLA-A68.1 HLA Type 300.000 Ser-Ser-Thr-Asp- N/A Arg (SEQ ID NO: 29)Arg-Arg-Lys-Asn- 4 HLA-B*08 20 HLA Type Tyr-Gly-Gln-Leu N/A (SEQ ID NO:11) Ser-Pro-Tyr-Glu- 44 HLA-B*08 20 HLA Type Lys-Val-Ser-Ala N/A (SEQ IDNO: 30) Ser-Pro-Tyr-Glu- 44 HLA-B*08 22 HLA Type Lys-Val-Ser-Ala- N/AGly (SEQ ID NO: 31) Cys-Arg-Arg-Lys- 3 HLA- 23 HLA Type Asn-Tyr-Gly-Gln-B*2705 N/A Leu (SEQ ID NO: 15) Gly-Arg-Tyr-Val- 30 HLA- 18 1000.000Pro-Pro-Ser-Ser- B*2705 Thr (SEQ ID NO: 32) Ala-Arg-Asp-Thr- 13 HLA- 12200.000 Tyr-His-Pro-Met- B*2705 Ser (SEQ ID NO: 33) Lys-Asn-Tyr-Gly- 6HLA- 17 150.000 Gln-Leu-Asp-Ile- B*2705 Phe (SEQ ID NO: 34)Gly-Gln-Leu-Asp- 9 HLA- 17 100.000 Ile-Phe-Pro-Ala- B*2705 Arg (SEQ IDNO: 35) Arg-Arg-Lys-Asn- 4 HLA- 14 60.000 Tyr-Gly-Gln-Leu- B*2705 Asp(SEQ ID NO: 36) Cys-Arg-Arg-Lys- 3 HLA- 21 HLA Type Asn-Tyr-Gly-Gln-B*2709 N/A Leu (SEQ ID NO: 15) His-Pro-Met-Ser- 21 HLA- HLA Type 60.000Glu-Tyr-Pro-Thr- B*3501 N/A Tyr (SEQ ID NO: 37) Ser-Ala-Gly-Asn- 50 HLA-HLA Type 60.500 Gly-Gly-Ser-Ser- B*5102 N/A Leu (SEQ ID NO: 5)Arg-Lys-Asn-Tyr- 5 HLA- 26 HLA Type Gly-Gln-Leu-Asp- DRB1*0101 N/AIle-Phe-Pro-Ala- Arg-Asp-Thr (SEQ ID NO: 38) Arg-Ser-Pro-Tyr- 43 HLA- 26HLA Type Glu-Lys-Val-Ser- DRB1*0101 N/A Ala-Gly-Asn-Gly- Gly-Ser-Ser(SEQ ID NO: 39) Leu-Asp-Ile-Phe- 11 HLA- 24 HLA Type Pro-Ala-Arg-Asp-DRB1*0101 N/A Thr-Tyr-His-Pro- Met-Ser-Glu (SEQ ID NO: 40)Tyr-Glu-Lys-Val- 46 HLA- 24 HLA Type Ser-Ala-Gly-Asn- DRB1*0101 N/AGly-Gly-Ser-Ser- Leu-Ser-Tyr (SEQ ID NO: 41) Gly-Arg-Tyr-Val- 33 HLA- 22HLA Type Pro-Pro-Ser-Ser- DRB1*0101 N/A Thr-Asp-Arg-Ser- Pro-Tyr-Glu(SEQ ID NO: 42) Gly-Ser-Ser-Leu- 55 HLA- 22 HLA Type Ser-Tyr-Thr-Asn-DRB1*0101 N/A Pro-Ala-Val-Ala- Ala-Ala-Ser (SEQ ID NO: 43)Tyr-Gly-Gln-Leu- 8 HLA- 20 HLA Type Asp-Ile-Phe-Pro- DRB1*0101 N/AAla-Arg-Asp-Thr- Tyr-His-Pro (SEQ ID NO: 44) Arg-Lys-Asn-Tyr- 5 HLA- 22HLA Type Gly-Gln-Leu-Asp- DRB1*0401 N/A Ile-Phe-Pro-Ala- Arg-Asp-Thr(SEQ ID NO: 38) Tyr-Gly-Gln-Leu- 8 HLA- 20 HLA Type Asp-Ile-Phe-Pro-DRB1*0401 N/A Ala-Arg-Asp-Thr- Tyr-His-Pro (SEQ ID NO: 44)Tyr-His-Pro-Met- 20 HLA- 20 HLA Type Ser-Glu-Tyr-Pro- DRB1*0401 N/AThr-Tyr-His-Thr- His-Gly-Arg (SEQ ID NO: 45) Gly-Arg-Tyr-Val- 33 HLA- 20HLA Type Pro-Pro-Ser-Ser- DRB1*0401 N/A Thr-Asp-Arg-Ser- Pro-Tyr-Glu(SEQ ID NO: 42) Tyr-Pro-Thr-Tyr- 26 HLA- 25 HLA Type His-Thr-His-Gly-DRB1*1101 N/A Arg-Tyr-Val-Pro- Pro-Ser-Ser (SEQ ID NO: 46)Arg-Lys-Asn-Tyr- 5 HLA- 22 HLA Type Gly-Gln-Leu-Asp- DRB1*1101 N/AIle-Phe-Pro-Ala- Arg-Asp-Thr (SEQ ID NO: 38) Arg-Ser-Pro-Tyr- 43 HLA- 22HLA Type Glu-Lys-Val-Ser- DRB1*1101 N/A Ala-Gly-Asn-Gly- Gly-Ser-Ser(SEQ ID NO: 39) Location is the position of the first residue. ¹Scoreobtained using program developed by Rammensee, et al. (1999) supra.²Score obtained using program developed by Parker, et al. (1994) supra.³Minimum scores of 20 on the SYFPEITHI site or 60 on the BIMAS site wereused. If a score met the requirement of one site, the score of the othersite is listed even if the minimum score for that site was not achieved.N/A is not available.

These results demonstrate that the cytoplasmic tail of MUC1, which is87% identical between humans and mice, may be utilized for methods ofimmunizing against MUC1-positive tumors in humans.

Accordingly, the efficacy of cytoplasmic tail peptide vaccination wasevaluated by challenging MUC1.Tg mice with a lethal dose ofMUC1-expressing B16 tumor cells 10 days following vaccination. MUC1.Tgmice vaccinated with MUC1 cytoplasmic tail peptides demonstrated asignificant increase in survival compared to MUC1.Tg mice vaccinatedwith control peptide or nonvaccinated mice (p<0.05). Organs expressingendogenous MUC1 were examined at necropsy and histologically todetermine whether the vaccination also elicited detectable autoimmunity.No evidence of autoimmunity was detected in any of the animals.

MUC1 expression in tumors was measured among vaccinated, controlpeptide-vaccinated and nonvaccinated mice. Serial sections of tumortissue were examined for expression of MUC1 by immunohistochemistry.Tumors from control peptide-vaccinated MUC1.Tg mice challenged withB16.MUC1 showed significantly greater expression of MUC1 than vaccinatedmice challenged with B16.MUC1. Similar results were observed in tumorsobtained from nonvaccinated mice challenged with B16.MUC1 compared tovaccinated mice challenged with B16.MUC1.

The efficacy of MUC1 cytoplasmic tail peptide vaccinations to protectagainst MUC1-expressing Panc02 tumors was evaluated by challengingMUC1.Tg mice with a lethal dose of Panc02.MUC1 tumor cells 10 daysfollowing the final vaccination. MUC1.Tg mice vaccinated with MUC1cytoplasmic tail peptideCys-Gln-Cys-Arg-Arg-Lys-Asn-Tyr-Gly-Gln-Leu-Asp-Ile-Phe-Pro-Ala-Arg-Asp-Thr-Tyr-His-Pro-Met-Ser-Glu-Tyr-Pro-Thr-Tyr-His(SEQ ID NO:47) demonstrated a significant increase in survival comparedto MUC1.Tg mice vaccinated with control peptide or nonvaccinated mice(p<0.05). Organs from these mice examined at necropsy and histologicallyshowed no evidence of autoimmunity.

Serial sections of tumor tissue derived from vaccinated, controlpeptide-vaccinated, and nonvaccinated mice were evaluated byimmunohistochemistry for expression of MUC1. Tumors from controlpeptide-vaccinated MUC1.Tg mice challenged with Panc02.MUC1 showedsignificantly more MUC1 expression than vaccinated mice challenged withPanc02.MUC1. Similar reactivity was observed in tumors obtained fromnonvaccinated mice challenged with Panc02.MUC1 compared to vaccinatedmice challenged with Panc02.MUC1. These results, combined with survivaldata, indicated that MUC1-expressing tumor cells were eliminated, andthat tumor growth was primarily due to MUC1-negative variants. Moreover,the observed anti-MUC1 immune responses were tumor specific, as therewas no evidence of autoimmune reactions.

To investigate whether observed anti-MUC1 immune responses were mediatedby humoral and/or cell-mediated mechanisms, ELISA and CTL assays wereperformed. For ELISA, sera from vaccinated, control peptide-vaccinated,and nonvaccinated mice challenged with MUC1-expressing B16 or Panc02tumors were tested for production of antibodies to a peptide of SEQ IDNO:47, peptideHis-Pro-Met-Ser-Glu-Tyr-Pro-Thr-Tyr-His-Thr-His-Gly-Arg-Tyr-Val-Pro-Pro-Ser-Ser-Thr-Asp-Arg-Ser-Pro-Tyr-Glu-Lys-Val-Ser-Ala-Gly(SEQ ID NO:48), peptideSer-Thr-Asp-Arg-Ser-Pro-Tyr-Glu-Lys-Val-Ser-Ala-Gly-Asn-Gly-Gly-Ser-Ser-Leu-Ser-Tyr-Thr-Asn-Pro-Ala-Val-Ala-Ala-Ala-Ser-Ala-Asn-Leu(SEQ ID NO:49), control peptide, or tandem repeat peptide. There were nostatistically significant differences in antibody responses to peptidesamong the vaccinated, control peptide-vaccinated and nonvaccinated mice(p>0.05) with one exception. Following challenge with MUC1-expressingPanc02 tumor cells, nonvaccinated mice produced significantly morepeptide-specific antibodies to peptides compared to vaccinated orcontrol peptide-vaccinated mice (p<0.05).

To evaluate generation of MUC1-specific CTL, spleens and lymph nodeswere harvested from vaccinated, control peptide-vaccinated andnonvaccinated MUC1.Tg mice challenged with MUC1-expressing B16 or Panc02tumors. MUC1-specific cytolytic activity was measured against EL4 orEL4.MUC1 cells. CTL were restimulated in vitro for 10 days with EL4 orEL4.MUC1 cells prior to analysis. Cytotoxic responses to EL4.MUC1 cellswere similarly low among control peptide-vaccinated and nonvaccinatedmice challenged with B16.MUC1 or Panc02.MUC1 (p>0.05). In contrast,lymphocytes obtained from vaccinated mice challenged with B16.MUC1 orPanc02.MUC1 demonstrated significant lysis of EL4.MUC1 cells (p<0.05).These responses were specific for MUC1 because there were no differencesof EL4 cell lytic activity among vaccinated, control peptide vaccinatedand nonvaccinated mice challenged with MUC1-expressing B16 or Panc02tumor cells (p>0.05).

It has now been demonstrated that portions of the cytoplasmic tail ofMUC1 mediate immunological rejection of MUC1-expressing tumor cells.Further, vaccination of immunologically tolerant MUC1.Tg mice withpeptides derived from the amino acid sequence of the MUC1 cytoplasmictail elicits a protective immune response that significantly prolongssurvival of these mice following challenge with MUC1-expressing B16 orPanc02 tumor cells.

Accordingly, one aspect of the present invention is a peptide of atleast a portion of a MUC1 cytoplasmic tail peptide of SEQ ID NO:1,wherein said peptide is useful for preventing or treating cancer. A MUC1cytoplasmic tail peptide which may be used within the scope of theinvention includes a full-length MUC1 cytoplasmic tail peptide (SEQ IDNO:1), or a homolog, an allele, an ortholog, or a portion of SEQ ID NO:1which induces an immune response to MUC1-expressing tumor cells.Preferably, the immune response is characterized by the elicitation of aT cell response (e.g., T helper or cytotoxic T cells) which is broughtabout by exposure to a MUC1 cytoplasmic tail peptide. More preferably,the MUC1 cytoplasmic tail peptide binds to a MHC class I or MHC class IImolecule or is an MHC non-restricted epitope thereby inducing an immuneresponse. In a preferred embodiment of the invention, a portion of aMUC1 cytoplasmic tail peptide encompasses a peptide of SEQ ID NO:47, SEQID NO:48 or SEQ ID NO:49. In a more preferred embodiment, a portion of aMUC1 cytoplasmic tail peptide is a 9 to 15 amino acid residue portion ofSEQ ID NO:1, e.g., peptides of SEQ ID NO:2 to SEQ ID NO:46.

A MUC1 cytoplasmic tail peptide of the invention may berecombinantly-produced or chemically-synthesized using conventionalmethods well-known to the skilled artisan.

In general, recombinant production of a MUC1 cytoplasmic tail peptidemay require incorporation of nucleic acid sequences encoding saidpeptide into a recombinant expression vector in a form suitable forexpression of the peptide in a host cell. A suitable form for expressionprovides that the recombinant expression vector includes one or moreregulatory sequences operatively-linked to the nucleic acids encodingthe a MUC1 cytoplasmic tail peptide in a manner which allows fortranscription of the nucleic acids into mRNA and translation of the mRNAinto the protein. Regulatory sequences may include promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Such regulatory sequences are known to those skilled in the art and aredescribed in Goeddel D. D., ed., Gene Expression Technology, AcademicPress, San Diego, Calif. (1991). It should be understood that the designof the expression vector may depend on such factors as the choice of thehost cell to be transfected and/or the level of expression required.Nucleic acid sequences or expression vectors harboring nucleic acidsequences encoding a MUC1 cytoplasmic tail peptide may be introducedinto a host cell, which may be of eukaryotic or prokaryotic origin, bystandard techniques for transforming cells. Suitable methods fortransforming host cells may be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor LaboratoryPress (2000)) and other laboratory manuals. The number of host cellstransformed with a nucleic acid sequence encoding a MUC1 cytoplasmictail peptide will depend, at least in part, upon the type of recombinantexpression vector used and the type of transformation technique used.Nucleic acids may be introduced into a host cell transiently, or moretypically, for long-term expression of a MUC1 cytoplasmic tail peptidethe nucleic acid sequence is stably integrated into the genome of thehost cell or remains as a stable episome in the host cell. Onceproduced, a MUC1 cytoplasmic tail peptide may be recovered from culturemedium as a secreted polypeptide, although it also may be recovered fromhost cell lysates when directly expressed without a secretory signal.When a MUC1 cytoplasmic tail peptide is expressed in a recombinant cellother than one of human origin, the MUC1 cytoplasmic tail peptide issubstantially free of proteins or polypeptides of human origin. However,it may be necessary to purify the MUC1 cytoplasmic tail peptide fromrecombinant cell proteins or polypeptides using conventional proteinpurification methods to obtain preparations that are substantiallyhomogeneous as to the MUC1 cytoplasmic tail peptide.

In addition to recombinant production, a MUC1 cytoplasmic tail peptidemay be produced by direct peptide synthesis using solid-phase techniques(Merrifield J: (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer, Boston, Mass.). Various fragments ofthe MUC1 cytoplasmic tail peptide may be chemically-synthesizedseparately and combined using chemical methods to produce a full-lengthmolecule.

Whether recombinantly-produced or chemically-synthesized, a MUC1cytoplasmic tail peptide or portion thereof may be further modifiedprior to use. For example, the peptides may be glycosylated,phosphorylated or fluorescently-tagged using well-known methods.

MUC1 cytoplasmic tail peptides of the invention are useful in inducingan immune response to MUC1-expressing tumor cells. Accordingly, anotheraspect of the present invention is a method for preventing or treatingcancer in a subject by administering a MUC1 cytoplasmic tail peptideprovided herein. Subjects who may benefit from a MUC1 cytoplasmic tailpeptide of the invention include those having, at risk of having, orsuspected of having cancer. A subject at risk of having cancer mayinclude individuals who have a high probability of developing cancer(e.g., individuals who have been exposed to cancer causing agents) orwho may have a genetic predisposition for developing cancer and maybenefit from a preventive therapy. Preferably, a subject has, is at riskof having or is suspected of having a cancer in which the tumor cellsexpress MUC1. Cancers which may be prevented or treated include cancersof secretory epithelia origin including, but not limited to, cancers ofthe pancreas, breast, prostate, liver, colon, and others.

An effective amount of MUC1 cytoplasmic tail peptide which may be usedin accordance with the method of the invention is an amount whichprevents, eliminates, alleviates, or reduces at least one sign orsymptom of a cancer. Signs or symptoms associated with a cancer that maybe monitored to determine the effectiveness of a MUC1 cytoplasmic tailpeptide include, but are not limited to, tumor size, feelings ofweakness, pain perception, and the like. The amount of the MUC1cytoplasmic tail peptide required to achieve the desired outcome ofpreventing, eliminating, alleviating or reducing a sign or symptom ofcancer will be dependent on the pharmaceutical composition of the MUC1cytoplasmic tail peptide, the patient and the condition of the patient,the mode of administration, and the type of cancer being prevented ortreated. For example, from about 0.05 μg to about 20 mg per kilogram ofbody weight per day of MUC1 cytoplasmic tail peptide may beadministered. Dosage regime may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may, beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A MUC1cytoplasmic tail peptide may be administered by continuous orintermittent infusion, parenterally, intramuscularly, subcutaneously,intravenously, intra-arterially, intrathecally, intraarticularly,transdermally, orally, bucally, intranasally, as a suppository orpessary, topically, as an aerosol, spray, or drops, depending uponwhether the preparation is used to treat an internal or external cancer.Such administration may be accompanied by pharmacologic studies todetermine the optimal dose and schedule and would be within the skill ofthe ordinary practitioner.

A pharmaceutical composition is one which contains a MUC1 cytoplasmictail peptide and a pharmaceutically acceptable carrier. Apharmaceutically acceptable carrier is a material useful for the purposeof administering the medicament, which is preferably sterile andnon-toxic, and may be solid, liquid, or gaseous materials, which isotherwise inert and medically acceptable, and is compatible with theactive ingredients. A generally recognized compendium of methods andingredients of pharmaceutical compositions is Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. LippincottWilliams & Wilkins: Philadelphia, Pa., 2000.

A pharmaceutical composition may contain other active ingredients suchas preservatives. A pharmaceutical composition may take the form of asolution, emulsion, suspension, ointment, cream, granule, powder, drops,spray, tablet, capsule, sachet, lozenge, ampoule, pessary, orsuppository. Further, a MUC1 cytoplasmic tail peptide may be coated by,or administered with, a material to prevent its inactivation. Forexample, peptides may be administered in an adjuvant, co-administeredwith enzyme inhibitors or in liposomes. Adjuvants contemplated hereininclude resorcinols, non-ionic surfactants such as polyoxyethylene oleylether and n-hexadecyl polyethylene ether. Enzyme inhibitors includepancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) andtrasylol. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes.

In a preferred embodiment, a MUC1 cytoplasmic tail peptide according tothe invention may be administered as a vaccine to cancer patients toinduce immunity to MUC1. Accordingly, a MUC1 cytoplasmic tail peptidemay be conjugated to a carrier protein such as, for example, tetanustoxoid, diphtheria toxoid or oxidized KLH in order to stimulate T cellhelp.

It is further contemplated that a MUC1 cytoplasmic tail peptide may beconjugated to other species. The other species comprehended include allchemical species which can be fused to the peptide without affecting thebinding of the peptide by T-cells. Specific examples are, for example,other antigens such as epitopes which may elicit a separate immuneresponse, carrier molecules which may aid absorption or protect thepeptide from enzyme action in order to improve the effective half-lifeof the peptide. For example, while it may be desirable to use a peptideof SEQ ID NO:47 or fragment thereof to treat or prevent pancreaticcancer, peptides or fragments of peptides of SEQ ID NO:47 in combinationwith peptides of the tandem repeat region of MUC1 may be useful intreating or preventing breast cancer.

Compositions and vaccines according to the invention may contain asingle MUC1 cytoplasmic tail peptide or a range of MUC1 cytoplasmic tailpeptides which cover different or similar epitopes. In addition oralternatively, a single polypeptide may be provided with multipleepitopes. The latter type of vaccine is referred to as a polyvalentvaccine.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Cell Culture

The BL6 variant of the C57BL/6-derived murine melanoma cell line B16 wasmaintained in Dulbecco's Minimal Essential Medium (DMEM) (GIBCO™ BRL,Div. of Life Technologies Inc., Rockville, Md.) supplemented with 10%heat-inactivated fetal bovine serum (BioWhittaker, Walkersville, Md.),essential amino acids (BioWhittaker), non-essential amino acids(Biowhittaker), sodium pyruvate (Sigma, St. Louis, Mo.), vitamins(GIBCO™), and penicillin/streptomycin (Biowhittaker). Panc02, amethylcholanthrene-induced pancreatic carcinoma syngeneic to C57BL/6,was maintained in McCoy's 5A medium (GIBCO™) supplemented with 10%heat-inactivated fetal bovine serum, and penicillin/streptomycin in ahumidified incubator at 37° C. and 5% CO₂. EL4 cells were cultured inRPMI 1640 medium (GIBCO™) supplemented with 10% heat-inactivated fetalbovine serum and penicillin/streptomycin in a humidified incubator at37° C. and 5% CO₂. Culture media for MUC1 transfectant clones of B16,Panc02 and EL4 were supplemented with 600 μg/mL G418 (Mediatech,Herndon, Va.).

Example 2 Expression of Epitope-Tagged MUC1 Deletion Constructs

B16 and Panc02 were transfected with plasmid DNA encoding a full-lengthhuman MUC1 cDNA (B16.MUC1 or Panc02.MUC1) or control expression vector(B16.neo, or Panc02.neo) as has been described (Rowse, et al. (1998)supra; Morikane, et al. (1999) supra). The full-length cytoplasmic tailwas 69 amino acids in length. MUC1 cytoplasmic tail deletion constructswere generated using well-established methods (Pemberton, et al. (1996)J. Biol. Chem. 271:2332; Burdick, et al. (1997) J. Biol. Chem.272:24198). The tandem repeat was comprised of a 20 amino acid sequence(Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr-Ala-Pro-Pro-Ala-His-Gly-Val-Thr-Ser-Ala;SEQ ID NO:50) that repeated 42 times. The MUC1 tandem repeat deleted(ΔTR) construct was generated using well-known methods (Pemberton, etal. (1996) supra; Burdick, et al. (1997) supra). FLAG® epitope-taggedhuman MUC1 cDNA constructs in which portions of the cytoplasmic tail(MUC1.CT3, MUC1.CT33, MUC1.CT45), or tandem repeat (MUC1(ΔTR)) of MUC1were deleted were subcloned into the expression vector pHβ-Apr1-neousing well-known methods (Pemberton, et al. (1996) supra; Burdick, etal. (1997) supra; Gunning, et al. (1987) Proc. Natl. Acad. Sci. U.S.A.84:4831). B16 and Panc02 were transfected with plasmid DNA using theLIPOFECTIN® method (GIBCO™) or the GENEPORTER™ 2 method (Gene TherapySystems, San Diego, Calif.). Cells were plated in 100 mm tissue culturedishes (Falcon Plastics, BD Labware, Franklin Lakes, N.J.) and grown- toapproximately 70% confluence. For the LIPOFECTIN® method, growth mediumwas removed, and the cells were washed with 1×PBS, and incubated 12hours in 5 mL of serum-free DMEM with 10 μL LIPOFECTIN® reagent and 10μg plasmid DNA linearized by ScaI digestion. After 48 hours, thetransfection medium was replaced with selection medium containing 600μg/mL G418. For the GENEPORTER™ 2 method, growth medium was removed, andthe cells were washed with 1×PBS, and then incubated 48 hours in 5 mL ofserum-free DMEM containing 42 μL GENEPORTER™ 2 reagent, 12 μg linearizedplasmid DNA linearized by ScaI digestion, and 300 μL DNA diluent B (GeneTherapy Systems). Transfection reactions of Panc02 cells usingGENEPORTER™ 2 also contained 50 μL transfection booster 3 (Gene TherapySystems). After 48 hours, the transfection medium was replaced withselection medium containing 600 μg/mL G418. After approximately 7-10days, single colonies were selected with cloning cylinders and expanded.Clonal cell lines were evaluated for co-expression of FLAG® epitope andMUC1 epitopes by western blotting with anti-FLAG® antibody (M2) (Sigma)and anti-MUC1 antibodies HMFG-2 or CT-2. Cell lines found to express theFLAG® epitope and MUC1 epitopes by western blotting, were evaluated forFLAG® epitope and MUC1 epitope surface expression by flow cytometryanalysis.

Example 3 Preparation of Cell Lysates

Cell lysates were prepared by scraping cells into 1 mL of lysis buffer(10 mM Tris, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1% TRITON®X-100) with a rubber cell scraper. Lysates were incubated on ice, for 30minutes and centrifuged at 4° C. for two minutes at 6,000 rpm to removecell debris. Supernatants were transferred to fresh tubes and proteincontent was determined using the BTO-RAD® protein assay (BIO-RAD®,Hercules, Calif.) with bovine serum albumin standards. Cell lysates werestored at −20° C.

Example 4 Immunoblotting

Cell lysates were resolved on 10% denaturing polyacrylamide gels (with3% polyacrylamide stacking gel), electrophoretically transferred topolyvinylpyrrolidone difluoride membranes, and blocked overnight inblotto (5% dry milk in 1× Tris-buffered saline (0.9% NaCl, 10 mM Tris,pH 7.4, 0.5% MgCl₂)). Primary antibodies were diluted 1:2000 in blotto.Incubations were for 1 hour at room temperature with light shaking,followed by three 10 minute washes with blotto. Alkalinephosphatase-conjugated goat anti-mouse secondary antibodies were diluted1:2500 in blotto, and incubations were for 1 hour at room temperaturewith light shaking. Following incubation with secondary antibody, themembranes were washed three times as above. Enhanced chemifluorescence(ECF) reagents were applied as per manufacturer instructions (AMERSHAM™Life Science LTD., Buckinghamshire, UK), and blots were visualized usinga Fujifilm LAS-1000 CCD imaging system (Fuji Film Co., Tokyo, JP).Analysis was performed using IR-LAS-1000 Lite V.1.1 software (Fuji).

Example 5 Flow Cytometry

Flow cytometric analysis of MUC1 surface expression on B16 and Panc02transfectants was performed as follows. Adherent cells were releasedfrom tissue culture flasks by treating with 0.05 mM trypsin and 1.5 mMEDTA in phosphate-buffered saline (PBS) for 5 minutes at 37° C. Allsubsequent steps were carried out on ice. The cells were resuspended inFACS medium (1×PBS, 0.2% BSA, 0.1% sodium azide) at a concentration of1×10⁶ cells/mL, and incubated with M2 antibody or anti-MUC1 tandemrepeat antibody (HMFG-2) for 20 minutes at 4° C. The cells were washedwith FACS medium and incubated with a phycoerythrin-conjugated (PE)rabbit anti-mouse secondary antibody (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) for 30 minutes at 4° C. The cellswere washed again and resuspended in FACS medium, followed by analysison a FACSCALIBUR™ (Becton Dickinson, Mountain View, Calif.). Analysiswas performed with CELLQUEST™ software (Becton Dickinson).

Example 6 Mice

Male and female wild-type C57BL/6 mice were purchased from the NationalCancer Institute (Frederick, Md.). Age-matched MUC1.Tg mice wereobtained using standard breeding methods.

Example 7 Tumor Challenge

On the day of tumor challenge, adherent control and MUC1-expressing B16tumor cell lines were released from tissue culture flasks by treatingwith 0.05 mM trypsin and 1.5 mM EDTA in PBS for 5 minutes at 37° C.,counted, and resuspended in DMEM at a concentration of 2×10⁵ viablecells/mL. Control and MUC1-expressing Panc02 tumor cell lines weresimilarly prepared except they were resuspended at 1×10⁷ viablecells/mL. 2×10⁴ viable B16.MUC1, B16.MUC1.CT3, B16.MUC1.CT33,B16.MUC1.CT45, B16.MUC1 (ΔTR), or B16.neo cells were injectedsubcutaneously, between the scapulae. 1×10⁶ viable Panc02.MUC1,Panc02.MUC1.CT3, Panc02.MUC1.CT33, Panc02.MUC1.CT45, Panc02.MUC1 (ΔTR),or Panc02.neo were injected subcutaneously, between the scapulae. Tumorgrowth was evaluated every two to three days, and tumor diameter wasmeasured using a caliper. Kaplan-Meier survival curves were prepared fortumor challenge studies. Death was defined as the date on which thetumor diameter measured 10 mm. Mice were euthanized when the tumordiameter exceeded 10 mm. The log-rank test was used for statisticalanalyses.

Example 8 Epitope Prediction

Two web-based algorithms were used to analyze the amino acid sequence ofMUC1 cytoplasmic tail for potential human and murine MHC class I andclass II binding epitopes. The first algorithm, “SYFPEITHI,” (Rammensee,et al (1999) supra) ranks peptides according to a score taking intoaccount the presence of primary and secondary MHC-binding anchorresidues. The second algorithm, “BIMAS,” (Parker, et al (1994) supra)ranks potential binding according to the predicted half-time ofdissociation of peptide/MHC complexes.

Example 9 Synthetic Peptides

Peptides used were:

Cys-Gin-Cys-Arg-Arg-Lys-Asn-Tyr-Gly-Gln-Leu-Asp-Ile-Phe-Pro-Ala-Arg-Asp-Thr-Tyr-His-Pro-Met-Ser-Glu-Tyr-Pro-Thr-Tyr-His(SEQ ID NO:47), a 30 amino acid peptide corresponding to MUC1cytoplasmic tail amino acid residues −3 to 27;

His-Pro-Met-Ser-Glu-Tyr-Pro-Thr-Tyr-His-Thr-His-Gly-Arg-Tyr-Val-Pro-Pro-Ser-Ser-Thr-Asp-Arg-Ser-Pro-Tyr-Glu-Lys-Val-Ser-Ala-Gly(SEQ ID NO:48), a 32 amino acid peptide corresponding to MUC1cytoplasmic tail amino acid residues 18 to 49, wherein the underlinedamino acid residues represent overlapping sequences with peptides of SEQID NO:47 and SEQ ID NO:49; and

Ser-Thr-Asp-Arg-Ser-Pro-Tyr-Glu-Lys-Val-Ser-Ala-Gly-Asn-Gly-Gly-Ser-Ser-Leu-Ser-Tyr-Thr-Asn-Pro-Ala-Val-Ala-Ala-Ala-Ser-Ala-Asn-Leu(SEQ ID NO:49), a 33 amino acid peptide corresponding to MUC1cytoplasmic tail amino acid residues 37 to 69.

Additional peptides used include:

His-Ser-Pro-Thr-Met-Asp-Arg-Ser-Glu-Ser-Tyr-Pro-Pro-Tyr-Thr-Glu-Tyr-Lys-His-Ser-Gly-Ala-Val(SEQ ID NO:51), a 23 amino acid peptide corresponding to the scrambledsequence of the overlapping regions peptides of SEQ ID NO:47, SEQ IDNO:48 and SEQ ID NO:49, referred to as control peptide; and

a 20 amino acid peptide containing one complete MUC1 tandem repeat(Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr-Ala-Pro-Pro-Ala-His-Gly-Val-Thr-Ser-Ala;SEQ ID NO:50), referred to as TR.

All peptides were synthesized, characterized, and purified to >95%purity by Genemed Synthesis, Inc. (South San Francisco, Calif.).

Example 10 MUC1 Cytoplasmic Tail peptide Vaccinations and TumorChallenge

Peptides were diluted in PBS, pH 7.4, at a concentration of 500 μg/mL.The mixture was vortexed vigorously for 5 minutes. Mice receivingB16.MUC1 tumors received one vaccination consisting of a combination of50 μg of peptide of SEQ ID NO:47, 50 μg of peptide of SEQ ID NO:48, and50 μg of peptide of SEQ ID NO:49 in a total volume of 100 μL by s.cinjection between the scapulae. Control mice received one vaccinationconsisting of 50 μg of control peptide in a total volume of 100 μL bys.c injection between the scapulae or no vaccination. Ten days followingvaccination, 2×10⁴ viable B16.MUC1 cells were injected subcutaneously,between the scapulae. Mice receiving Panc02.MUC1 tumors received threevaccinations, at seven day intervals, consisting of 50 μg of peptide ofSEQ ID NO:47 in a total volume of 100 μL by s.c injection between thescapulae. Control mice received three vaccinations, at seven dayintervals, consisting of 50 μg of control peptide in a total volume of100 μL by s.c injection between the scapulae or no vaccination. Ten daysfollowing the third vaccination, 1×10⁶ viable Panc02.MUC1 cells wereinjected subcutaneously, between the scapulae. Tumor challenge resultswere evaluated as described herein.

Example 11 Immunohistochemical Examination of Tumors

For immunohistochemical evaluation of MUC1 expression on tumors obtainedfrom vaccinated, control-vaccinated, and nonvaccinated animals, tumortissue was fixed in a buffered formalin solution (100 mL formalin, 3.4 gNaH₂PO₄, and 10.3 g Na₂HPO₄)/1000 mL, pH 7.3-7.4, embedded in paraffinand sectioned at a thickness of 5 μm. Tissue sections were assayed usinga modification of an ABC immunohistochemical method (Hsu, et al. (1981)J. Histochem. Cytochem. 29:577). Briefly, tissue sections weredeparaffinized in EZ-DEWAX™ (BioGenex, San Ramon, Calif.). Antigenunmasking was carried out by adding 10 mM citrate buffer and boiling for10 minutes. The sections were then incubated in blocking serum for 20minutes and primary monoclonal antibody M2 (Sigma) was added and thesamples were kept in a humid chamber at 4° C. overnight. The slides wererinsed with PBS and incubated for 1 hour with biotin-labeled secondarymonoclonal antibody. Endogenous peroxidase activity was blocked byincubating the samples in 3% H₂O₂ for 5 minutes. The slides were thenincubated for 30 minutes at room temperature with ABC reagent (VectorLabs, Burlingame, Calif.). The slides were then rinsed with PBS andincubated for 3-5 minutes with DAB substrate (Vector Labs) observingclosely for color to develop. Sections were then incubated for 10minutes in 50 mM sodium bicarbonate pH 9.6 followed by a 5-20 secondincubation in DAB enhancing solution (Vector Labs) and counterstainedwith Meyer's hematoxylin for 30 seconds. Cover slips were applied andthe slides were examined under a Nikon E400 microscope (Nikon, Tokyo,JP). Images were captured using a Nikon CoolPix 950 digital camera(Nikon).

Example 12 Cytotoxic T Lymphocyte Assay

Detection of MUC1-specific cytotoxic T lymphocytes (CTL) was carried outby generation of effector cells from spleens and lymph nodes (axillary,brachial, inguinal and mesenteric) harvested from vaccinated, controlpeptide-vaccinated and nonvaccinated mice. Red blood cells were lysedusing RBC lysis buffer (800 mg NH₄Cl, 80 mg EDTA, 80 mg NaHCO₃)/100 mL.EL4 and EL4.MUC1 target cells were labeled with 100 μCi Na₂ ⁵¹CrO₄ for1.5 hours and washed. Effector cells were incubated with 5×10³⁵¹Cr-labeled EL4 or EL4.MUC1 cells at various effector to target ratios(E:T) in triplicate in 96-well plates (Falcon) in a total volume of 200μL. The plates were incubated for 6 hours at 37° C. and 5% CO₂.Following incubation, 50 μL culture supernatant was transferred toLUMAPLATES™ (Packard Instrument Company, Inc, Meriden, Conn.). TheLUMAPLATES™ were air dried overnight at room temperature andradioactivity was measured using a TOPCOUNT® NXT (Packard). Percentspecific lysis was determined using the following equation:((experimental ⁵¹Cr release−spontaneous ⁵¹Cr release)/(maximum ⁵¹Crrelease−spontaneous ⁵¹Cr release))×100%=% specific lysis. Experimental⁵¹Cr release represents ⁵¹Cr release from targets mixed with effectors,spontaneous ⁵¹Cr release represents targets in medium only, and maximum⁵¹Cr release represents targets lysed with 5% TRITON® X-100.

Example 13 Statistical Analysis

Tumor challenge studies were conducted two or more times. Survival datawere pooled and the log-rank test was used for statistical analysis ofsurvival. Additionally, the Cox regression analysis was used to confirmstatistical differences among experimental groups and to verify thatdata from repeated experiments was statistically similar prior to beingpooled. For CTL assays, statistical significance was determined byone-way ANOVA followed by Newman-Keuls Multiple Comparison Test. For allstatistical tests, a p value <0.05 was considered to be statisticallysignificant.

1. A composition comprising an isolated MUC1 cytoplasmic tail peptideconsisting of the sequence selected from the group consisting of SEQ IDNO: 47, SEQ ID NO: 48, and SEQ ID NO:
 49. 2. The composition of claim 1,wherein said sequence of the MUC1 cytoplasmic tail peptide is SEQ ID NO:47.
 3. The composition of claim 1, wherein the composition comprises theMUC1 cytoplasmic tail peptide of SEQ ID NO: 47, the MUC1 cytoplasmictail peptide of SEQ ID NO: 48, and the MUC1 cytoplasmic tail peptide ofSEQ ID NO:
 49. 4. Q composition comprising MUC1 cytoplasmic tail peptideconsisting of the sequence selected from the group consisting of SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 20, SEQID NO: 23, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 44.